Community-based Study On Molecular Epidemiology Of Hepatitis B Virus In Zhaodong, Heilongjiang Province

Objective: To determine the distribution of hepatitis B virus (HBV) genotypes and serotypes in community-based Zhaodong, Heilongjiang province and to evaluate the relationship between genotypes and serotypes; To analyze the spectra of mutations of the S domin of HBV; To study the relationship between the genotypes and the outcomes of clinical patients.Methods: In 2005, 2 villages Shuangsheng and Xianfeng in Zhaodong, Heilongjiang province were selected by cluster-random design and vein blood were collected in all people. HBsAg,anti-HBs and anti-HBc were tested by SPRIA with kits. HBeAg and anti-HBe were measured by ELISA test kit. According to the serum epidemiology data of 1986 and the standard of"Chronic Hepatitis B virus prevention and therapy guid", together with the results of ALT, B-ultrasonic and physical examination, 110 sera samples were selected by clinical diagnosis. 86 sera samples were from asymptomatic HBV carriers (ASC), 12 from chronic hepatitis (CH), 5 from liver cirrhosis (LC), 1 from hepatocellular carcinoma (HCC) and 6 from convalescene. The mean age was 36 years. 60 were Male and 50 were female. Nested polymerase chain reaction was used for amplication of HBV DNA. HBV DNA was extracted from 200μl serum by QIAamp DNA Blood Mini Kit, according to the manufacture's recommendations. Two pairs of oligonucletide primers were used to amplify HBV DNA. The PCR products were subjected to electrophoresis on 1% agarose gel and visualized by using an ultraviolet transalluminator. The PCR products were purified using the Wizard SV Gel and PCR Clean-up System kit. DNA sequencing was performed by a commercial company using the ABI 3773 DNA System. The nucleotide sequences were compared with reference sequence of each genotype of A-H obtained from Genbank. Sequences were aligned, and phylogenetic tree was established using the DNASTAR software to determine the genotype. Serotype was determined by the deduced amino acid sequence from nucleotide sequence. Statistical analyses were performed using Fisher's exact test with SAS 8.1 software. P<0.05 was considered statistically significant.Result: Overall, HBV DNA was detectable in 97 (88.2%) of the 110 samples. A total of 97 samples of the S genes of HBV were analysed in our study. Genotype B was 9 (9.3%), C was 87 (89.7%) and D was 1 (1.0%). Adr was 77 (79.4%), adw2 was 2 (2.3%), ayr was 8 (9.2%) and ayw2 was 1 (1.0%). 9 genotype B all were adw2. In 87 genotype C, there were adrq+ 75 (86.2%), adrq- 2 (2.3%), adw2 2 (2.3%) and ayr 8 (9.2%). 1 genotype D was ayw2 (100%). There was a statistically significance among genotypes and serotypes (P=0.0000).Among 9 cases with genotype B,4 cases (44.4%) were ASC and 5 (55.6%) were CH. Among 87 cases with genotype C, 3 cases (3.4%) were convalescence, 73 (83.9%) ASC, 3 (3.4%) LC and 1 (1.2%) was HCC. 1 cases with genotype D was ASC. The clinical outcomes were significant different among all genotypes (P=0.0028).Comparing the 84 nucleotide sequences and the deduced amino acid sequences of the samples with the reference sequence in the Genbank database, we found 131 had substitutions, 40 had samesense mutations and 89 had missense mutations and 2 had nonsense mutations. In the major hydrophilic region (MHR, aa100-160), there were 45 substitutions. 18 were samesense mutations and 27 were missense mutations. There were 2 substitutions led to sense mutations Q129H (1) and M133L (1). The changes concentrated in the first loop of the'a'determinant. There was an insertion of three amino acids TTE in the aa119~120 site. The frequency of nucleotide substitution of S gene was 2.19/100nt.Conclusion:1 The predominant genotype of HBV in community-based Zhaodong, Heilongjiang province was genotype C, and the second common was genotype B, then genotype D.2 Serotype adr predominated (adrq+ and adrq- were both found, and adrq+ was more than adrq-) and had other serotypes such as adw2, ayr and ayw2. 3 Different genotypes had different serotypes. Most of genotype B were adw2, and most of genotype C were adr and others were adw2 and ayr, and genotype D was ayw2, which revealed that genotype and serotype had certain relationship.4. Genotype B was found in ASC and CH. Patients with genotype B were CH and ASC. Genotype C was found in ASC, CH, LC, HCC and convalescence, but most patients with genotype C were ASC. The ratio of genotype B in CH was increased, which revealed that patients with genotype B were easier to be CH. Most of the patients with genotype C were ASC.5. Although there were many nucleotide substitutions of S gene, about 1/3 were samesense mutations. There were mutations of S gene in the community people and ccurred in the first loop. Vaccine-associated mutation was not found. The frequency of nucleotide substitution of community-based HBV S gene was low, which revealed that S gene was relatively conserved.